The burgeoning size of the liver transplant waiting list has forced clinicians to accept more marginal donor organs, which have a higher susceptibility to preservation injury and graft dysfunction. The dysfunction is related, in part, to the invariable cold ischemia and reperfusion (CIR) injury associated with current organ transplantation techniques. Elevated mitochondrial calcium is a significant factor in hepatocellular cold ischemic injury and is associated with cytochrome c release and initiation of cellular apoptotic cascades. We have recently described the presence of two mitochondrial P2Y-like purinergic receptors, mP2Yi-like and mP2Y2-like, that increase mitochondrial Ca2+ uptake in response to changes in cytosolic [ATP]. The presence of mitochondrial purinergic receptors represents a novel mechanism regulating mitochondrial Ca2+ in CIR injury. The hypothesis of this application is that altered mitochondrial P2Y-like receptor stimulation in cold ischemia causes increased mitochondrial Ca2+ uptake and cytochrome c release that results in the activation of pro-apoptotic pathways. To investigate this hypothesis, we propose 3 specific aims. In specific aim 1. we will measure mitochondrial 45Ca2+ uptake in response to established agonists and inhibitors of plasma membrane P2Y1 and P2Y2 receptors and define the pharmacological profiles of mitochondrial P2Y-like receptors in isolated rat liver mitochondria. In specific aim 2. we will simultaneously stimulate mitochondrial P2Y-like receptors and antagonize mitochondrial Ca2+ management pathways to define the mechanism by which these receptors alter mitochondrial Ca2+ uptake and cytochrome c release. In specific aim 3, we will study HepG2 cells transfected with either GFP-tagged cytochrome c or recombinant, cell-compartment directed luciferase reporter genes in an in vitro model of CIR. We will establish the spatial-temporal relationship between cellular Ca movement and subcellular distribution of cytochrome c as a result of CIR using fluorometry and confocal microscopy. These data will be correlated with changes in mitochondrial and cytosolic [ATP] and cellular apoptosis. We will use caged purine analogs and receptor antagonists to control cytoplasmic concentrations and directly asses the impact of mP2Y1-like and mP2Y2-like receptor activity in CIR. The proposed research will elucidate a novel pathway involved in CIR injury and may lead to new treatment strategies to reduce CIR injury and graft dysfunction following liver transplantation.